This application address broad Challenge Area (06) Enabling Technologies and specific Challenge Topic (06-HL-103) on "Developing new imaging methodologies to track cells and measure accurately the chemical activities of enzymes and metabolites in intact cells, tissues, and organisms to improve basic understanding of cellular interactions, biological pathways, and their regulation". Imaging cell fate after transplantation is a high priority in both basic research and clinical translation. In order for cell based therapy to truly succeed, we must be able to track the location(s) of delivered cells, the duration of cell survival, and any potential adverse effects. Over the past 5 years, I have pioneered the reporter gene technology for studying the in vivo biology of cardiovascular stem cell therapy. This NIH Challenge Grant proposal represents a natural continuation of my dedicated efforts to push imaging of stem cell therapy from small animal models to pre-clinical large animals. In particular, our group has shown that the positron emission tomography (PET) reporter gene (PRG) and PET reporter probe (PRP) can be used to track gene transfer and cell delivery in rodent and porcine models. Here we would like to perform pre-clinical validation of the imaging of human cardiac resident stem cells in the next 2 years in step with the similar exciting progress that has been made in cancer stem cell imaging. Public Health Relevance: Coronary artery disease (CAD) is a progressive disease with high morbidity and mortality rates in the US. Following myocardial infarction (MI), the limited ability of the surviving cardiac cells to proliferate renders the damaged heart susceptible to unfavorable remodeling processes and morbid sequelae such as heart failure. For now, heart transplantation is the only viable treatment option for end-stage heart failure patients. Given the persistent shortage of donor heart organs, stem cell therapy has emerged as a promising candidate for treating ischemic heart disease because it provides a virtually unlimited source of cardiomyocytes, endothelial cells, and other differentiated cell types to be used in all stages of cardiac repair. Thus, imaging cell fate after transplantation is a high priority in both basic research and clinical translation. In order for cell based therapy to truly succeed, we must be able to track the location(s) of delivered cells, the duration of cell survival, and any potential adverse effects. Our major goals for this NIH Challenge Grant are the following: (1) To use versatile reporter gene constructs to understand the biology and physiology of cardiac stem cells (CSCs) in vivo and (2) To track cell fate in pre-clinical large animal models using combined positron emission tomography and computed tomography (PET/CT) imaging. Importantly, the information we obtain here on detection sensitivity and the overall experience we gain will be extremely valuable for eventual clinical translation of cardiac molecular imaging.